Anti-hypersensitive N2-tetrazole-β-glucuronide analog

ABSTRACT

Fermentation of the microorganism Streptomyces sp. MA6751 in the presence of   &lt;IMAGE&gt;   yields an anti-hypertensive compound of the structure   &lt;IMAGE&gt;

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel process for the preparation of theantihypertensive agent, compound (I) ##STR3## comprising fermentation ofcompound (II) ##STR4## with the microorganism Streptomyces sp. MA6751.Compound (I) and its precursor (II) are Angiotensin II (A II) receptorantagonists useful in the treatment of human hypertensive diseases.

A II is an octapeptide hormone produced mainly in the blood during thecleavage of Angiotensin I by angiotensin converting enzyme (ACE). ACE islocalized on the endothelium of blood vessels of lung, kidney and manyother organs. A II is the end product of the renin-angiotensin system(RAS), a system that plays a central role in the regulation of normalblood pressure and seems to be critically involved in hypertensiondevelopment and maintenance, as well as congestive heart failure. A IIis a powerful arterial vasconstrictor that exerts its action byinteracting with specific receptors present on cell membranes. A IIreceptor antagonism is one of the possible modes of controlling the RAS.

2. Brief Description of Disclosures in the Art

Copending U.S. application Ser. No. 516,286, filed May 4, 1990,discloses the substrate compound used in this invention and designatedherein as compound (II).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an ¹ H nuclear magnetic resonance (NMR) spectrum taken at 400MHz of compound (I) in CD₃ OD.

FIG. 2 is an ¹ H NMR spectrum taken at 400 NMz of compound (II) in CD₃OD.

SUMMARY OF THE INVENTION

A new antihypertensive biotransformation product (I) is obtained by thefermentation of the microorganism Streptomyces sp. A 6751, in thepresence of substrate compound (II). The biotransformation isaccomplished under submerged aerobic conditions in an aqueouscarbohydrate medium containing a nitrogen nutrient at a pH of about 7for a sufficient time to produce compound (I).

The resultant N2-tetrazole β-glucuronide analog exhibits A II antagonistactivity, i.e., it has an inhibitory concentration (IC₅₀) of less than50 μm in an assay that measures the concentration of potential A IIantagonist needed to achieve 50% displacement of the total ¹²⁵ I-Sar¹Ile⁸ -angiotensin II specifically bound to a membrane preparationderived from rabbit aortae, and therefore it is an antihypertensiveagent. This assay is also referred to herein as the "IC₅₀ " assay.

Also provided by this invention is a pharmaceutical compositioncontaining a therapeutically effective amount of compound (I) incombination with a pharmaceutically acceptable non-toxic carrier orexcipient.

In addition, there is provided a method of treating a human host tocontrol hypertensive disorders or for treating congestive heart failurecomprising administering to said host a therapeutically effective amountof compound (I).

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The product of this invention is compound (I): ##STR5##

The novel process of this invention comprises fermentation of themicroorganism Streptomyces sp. MA6751 in the presence of substratecompound (II) ##STR6## in a nutrient medium, and isolation of theresulting biotransformation product, compound (I), in a conventionalmanner. A biologically pure sample of Streptomyces sp. MA6751 iscurrently available in the permanent culture collection of the AmericanType Culture Collection, 12301 Parklawn Drive in Rockville, Maryland,from which it is available under the Accession Number ATCC 55043.

The following is a general description of Streptomyces sp. strainMA6751. Observations of growth, general cultural characteristics andcarbon source utilization were made in accordance with the methods ofShirling and Gottleib (Internat. J. System. Bacteriol. 16: 313-340).Chemical composition of the cells was determined using the methods ofLechevalier and Lechevalier (in Actinomycete Taxonomy. A. Dietz and D.W. Thayer, Ed. Society of Industrial Microbiology, 1980). Coloration ofthe culture was determined by comparison with color standards containedin the Inter-Society color Council-National Bureau of Standards CentroidColor Charts (U.S. Dept. of Commerce National Bureau of Standardssupplement to NBS Circular 553, 1985).

Source

The culture was isolated from soil at Heather forest, 9000-13,400 ft.elevation. MT. Kilimanjaro, Kenya.

Analysis of Cell Wall Composition

Peptidoglycan contains L-diaminopimelic acid. Whole cell analysis ofcarbohydrates reveals galactose, glucose, mannose and xylose.

General Growth Characteristics

Good growth on yeast malt extract, (w/o trace elements), glycerolasparagine, inorganic salts-starch, oatmeal, sporulation, and trypticasesoy agars. Growth occurs at 27° and 37° C. Culture also grows well inliquid media such as yeast dextrose broth.

Colony Morphology (on Yeast Malt Extract Agar)

Substrate mycelium is orange (71 m.OY) and colonies are opaque, raised,lobate and rubbery. The colony surface is rough textured. Aerial myceliaappear after 4 days incubation and are white in color (263 White). Sporemass, when present, is white (263 White).

Micromorphology

Aerial mycelium (0.57-0.76 μm dia.) arises from a substrate mycelium andis branched and flexous. In mature cultures, the aerial myceliumcommonly terminates in spores borne predominantly in spirals and flexouschains.

Miscellaneous Physiological Reactions

Culture does not produce melanoid pigments in tryptone yeast extractbroth, and yellow, non-pH dependant diffusible pigment is produced onglycerol asparagine agar. Starch is hydrolzed.

    ______________________________________                                        Cultural Characteristics of Streptomyces sp. MA6751 at 21 Days                                  Aerial                                                                        Mycelium                                                             Amount   and/or     Soluble Reverse                                  Medium   Growth   Spores     Pigments                                                                              Color                                    ______________________________________                                        Yeast Ex-                                                                              good     White (263),                                                                             none noted                                                                            Yellow                                   tract-            spiral             (s.Y 87)                                 Malt              sporophores                                                 Extract                                                                       Glucose- good     White (263),                                                                             Yellow, Yellow                                   Asparagine        spiral     non-pH  (p.Y 89)                                                   sporophores                                                                              dependent                                        Inorganic                                                                              good     White (263),                                                                             none noted                                                                            Yellow                                   Salts-Starch      spiral             (p.Y 89)                                                   sporophores                                                 Oatmeal  good     White (263),                                                                             none noted                                                                            Yellow                                                     spiral             (m.OY 84)                                                  sporophores                                                 ______________________________________                                    

    ______________________________________                                        Carbohydrate Utilization Pattern of Streptomyces sp.                          MA6751 at 21 Days                                                             Carbon Source    Utilization*                                                 ______________________________________                                        D-arabinose      0                                                            L-arabinose      1                                                            cellobiose       2                                                            D-fructose       2                                                            inositol         2                                                            α-D-lactose                                                                              2                                                            β-D-lactose 2                                                            D-maltose        2                                                            D-mannitol       3                                                            D-raffinose      2                                                            L-rhamnose       2                                                            sucrose          2                                                            D-xylose         2                                                            L-xylose         0                                                            α-D-glucose (control)                                                                    3                                                            ______________________________________                                         *3 = good utilization                                                         2 = moderate utilization                                                      1 = poor utilization                                                          0 = no utilization                                                       

In general, compound (I) can be produced by culturing (fermenting) theabove-described microorganism in the presence of an appropriateconcentration of substrate compound (II) in an aqueous nutrient mediumcontaining sources of assimilable carbon and nitrogen, preferably undersubmerged aerobic conditions (e.g., shaking culture, submerged culture,etc.). An appropriate concentration of the parent compound in theaqueous medium ranges from 0.05 mg/ml to 0.2 mg/ml, preferably 0.1mg/ml; less than 0.05 mg/ml is inefficient and greater than 0.2 mg/mlcan inhibit the culture. The aqueous medium is incubated at atemperature between 26° C. and 29° C., preferably 27° C.; culture growthwill be inhibited below this temperature range and culture death willoccur above this temperature range. The aqueous medium is incubated fora period of time necessary to complete the biotransformation asmonitored by HPLC, usually for a period of about 24 hours, on a rotaryshaker operating at about 220 rpm. with a throw of about 2 in. Theaqueous medium is maintained at a pH between 6 and 8, preferably about7, at the initiation and termination (harvest) of the fermentationprocess. A higher or lower pH will cause the culture to die. The desiredpH may be maintained by the use of a buffer such asmorpholinoethanesulfonic acid (MES), morpholino-propanesulfonic acid(MOPS), and the like, or by choice of nutrient materials whichinherently possess buffering properties, such as production mediadescribed herein below.

The preferred sources of carbon in the nutrient medium are certaincarbohydrates such as glucose, xylose, galactose, glycerin, starch,dextrin, and the like. Other sources which may be included are maltose,rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, andthe like.

The preferred sources of nitrogen are yeast extract, meat extract,peptone, gluten meal, cottonseed meal, soybean meal and other vegetablemeals (partially or totally defatted), casein hydrolysates, soybeanhydrolysates and yeast hydrolysates, corn steep liquor, dried yeast,wheat germ, feather meal, peanut powder, distiller's solubles, etc., aswell as inorganic and organic nitrogen compounds such as ammonium salts(e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.),urea, amino acids, and the like.

The carbon and nitrogen sources, though advantageously employed incombination, need not be used in their pure form because less purematerials which contain traces of growth factors and considerablequantities of mineral nutrients are also suitable for use. When desired,there may be added to the medium mineral salts such as sodium or calciumcarbonate, sodium or potassium phosphate, sodium or potassium chloride,sodium or potassium iodide, magnesium salts, copper salts, cobalt salts,and the like. If necessary, especially when the culture medium foamsseriously, a defoaming agent, such as liquid paraffin, fatty oil, plantoil, mineral oil or silicone may be added.

Submerged aerobic cultural conditions are preferred for the productionof compound (I) in massive amounts. For the production in small amounts,a shaking or surface culture in a flask or bottle is employed.Furthermore, when the growth is carried out in large tanks, it ispreferable to use the vegetative form of the organism for inoculation inthe production tanks in order to avoid growth lag in the process ofproduction of compound (I). Accordingly, it is desirable first toproduce a vegetative inoculum of the organism by inoculating arelatively small quantity of culture medium with spores or mycelia ofthe organism product in a "slant" and culturing said inoculated medium,also called the "seed medium", and then to transfer the culturedvegetative inoculum aspetically to large tanks. The fermentation medium,in which the inoculum is produced, is substantially the same as ordifferent from the medium utilized for the production of compound (I)and is generally autoclaved to sterilize the medium prior toinoculation. The fermentation medium is generally adjusted to a pHbetween 6 and 8, preferably about 7, prior to the autoclaving step bysuitable addition of an acid or base, preferably in the form of abuffering solution. Temperature of the seed medium is maintained between26° C. and 29° C., preferably 27° C.; culture growth will be inhibitedbelow this range and culture death will occur above this range.Incubation of the seed medium is usually conducted for a period of about10 to 30 hours, preferably 24 hours, on a rotary shaker operating at 220rpm; the length of incubation time may be varied according tofermentation conditions and scales. Agitation and aeration of theculture mixture maybe accomplished in a variety of ways. Agitation maybe provided by a propeller or similar mechanical agitation equipment, byrevolving or shaking the fermentor, by various pumping equipment or bythe passage of sterile air through the medium. Aeration may be effectedby passing sterile air through the fermentation mixture.

Preferred culturing/production media for carrying out the fermentationinclude the following media:

    ______________________________________                                                          g/l                                                         ______________________________________                                        Seed Medium A                                                                 Dextrose            1.0                                                       Dextrin             10.0                                                      Beef Extract        3.0                                                       Ardamine pH         5.0                                                       NZ Amine Type E     5.0                                                       MgSO.sub.4.7H.sub.2 O                                                                             0.05                                                      K.sub.2 HPO.sub.4   0.3                                                       Adjust pH to 7.1                                                              Add CaCO.sub.3 0.5 g/l                                                        Transformation Medium B                                                       Glucose             10                                                        Hycase SF           2                                                         Beef Extract        1                                                         Corn Steep Liquor   3                                                         Adjust pH to 7.0                                                              ______________________________________                                    

The product, compound (I), can be recovered from the culture medium byconventional means which are commonly used for the recovery of otherknown biologically active substances. Compound (I) is found in thecultured mycelium and filtrate, which are obtained by filtering orcentrifuging the cultured broth, and accordingly can be isolated andpurified from the mycelium and the filtrate by a conventional methodsuch as concentration under reduced pressure, lyophilization, extractionwith a conventional solvent, such as methylene chloride and the like, pHadjustment, treatment with a conventional resin (e.g. anion or cationexchange resin, non-ionic adsorption resin, etc.), treatment with aconventional adsorbent (e.g. activated charcoal, silicic acid, silicagel, cellulose, alumina, etc.), crystallization, recrystallization, andthe like. A preferred recovery method is solvent extraction,particularly using methylene chloride. A preferred purification methodinvolves the use of chromatography, especially high performance liquidchromatography (HPLC), using a silica gel column and an eluant mixturecomposed of water and an organic solvent such as methanol, acetonitrileand the like, and a small amount of acid such as acetic acid,trifluoracetic acid, phosphoric acid and the like. A preferred eluant iscomposed of acetonitrile and water containing 0.1% trifluoroacetic acid,and is run through the column in a linear gradient.

The compound of this invention forms salts with various inorganic andorganic acids and bases which are also within the scope of theinvention. Such salts include ammonium salts, alkali metal salts likesodium and potassium salts, alkaline earth metal salts like the calciumand magnesium salts, salts with organic bases, e.g., dicyclohexylaminesalts, N-methyl-D-glucamine, salts with amino acids like arginine,lysine and the like. Also, salts with organic and inorganic acids may beprepared, e.g., HCl, HBr, H₂ SO₄, H₃ PO₄, methane-sulfonictoluene-sulfonic, maleic, fumaric, camphorsulphonic. The non-toxic,physiologically acceptable salts are preferred.

The salts can be formed by conventional means such as by reacting thefree acid or free base forms of the product with one or more equivalentsof the appropriate acid or base in a solvent or medium in which the saltis insoluble, or in a solvent such as water which is then removed undervacuum or by freeze-drying, or by exchanging the cations of an existingsalt for another cation on a suitable ion exchange resin.

Suitable formulations of the material may also include conventionalpharmaceutically acceptable biolabile ester of compounds (I) formed viathe hydroxy groups or carboxy group on the molecule, such as theacetate.

The product of this invention, compound (I), exhibits A II antagonistactivity by the IC₅₀ assay, and therefore is useful in treatinghypertension. It is also of value in the management of acute and chroniccongestive heart failure. This compound may also be expected to beuseful in the treatment of secondary hyperaldosteronism, primary andsecondary pulmonary hyperaldosteronism, primary and secondary pulmonaryhypertension, renal failure such as diabetic nephropathy,glomerulonephritis. scleroderma, glomerular sclerosis, proteinuria ofprimary renal disease, end stage renal disease, renal transplanttherapy, and the like, renal vascular hypertension, left ventriculardysfunction, diabetic retinopathy and in the management of vasculardisorders such as migraine, Raynaud's disease, luminal hyperplasia, andto minimize the atherosclerotic process. The product of this inventionis also useful for cognitive function enhancement. The application ofthe compound of this invention for these and similar disorders will beapparent to those skilled in the art.

The compound of this invention is also useful to treat elevatedintraocular pressure and to enhance retinal blood flow and can beadministered to patients in need of such treatment with typicalpharmaceutical formulations such as tablets, capsules, injectables, aswell as topical ocular formulations in the form of solutions, ointments,inserts, gels, and the like. Pharmaceutical formulations prepared totreat intraocular pressure would typically contain about 0.1 to 15% byweight, preferably 0.5% to 2% by weight, of the compound of thisinvention.

In the management of hypertension and the clinical conditions notedabove, the compound of this invention may be utilized in compositionssuch as tablets, capsules or elixirs for oral administration,suppositories for rectal administration, sterile solutions or suspensionfor parenteral or intramuscular administration, and the like. Thecompounds of this invention can be administered to patients (animals andhuman) in need of such treatment in dosages that will provide optimalpharmaceutical efficacy, Although the dose will vary from patient topatient depending upon the nature and severity of disease, the patient'sweight, special diets then being followed by a patient, concurrentmedication, and other factors which those skilled in the art willrecognize, the dosage range will generally be about 1 to 1000 mg. perpatient per day which can be administered in single or multiple doses.Perferably, the dosage range will be about 2.5 to 250 mg. per patientper day; more preferably about 2.5 to 75 mg. per patient per day.

The compound of this invention can also be administered in combinationwith other antihypertensives such as α-methyldopa, and/or diuretics suchas hydrochlorothiazide, and/or antiotensin converting enzyme inhibitorssuch as enalapril, and/or calcium channel blockers such as nifedipine.Typically, the individual daily dosages for these combinations can rangefrom about one-fifth of the minimally recommended clinical dosages tothe maximum recommended levels for the entities when they are givensingly. These dose ranges can be adjusted on a unit basis as necessaryto permit divided daily dosage and, and as noted above, the dose willvary depending on the nature and severity of the disease, weight of thepatient, special diets and other factors. Typically, these combinationscan be formulated into pharmaceutical compositions as discussed below.

About 1 to 100 mg. of compound (I) of a physiologically acceptable saltthereof is compounded with a physiologically acceptable vehicle,carrier, excipient, binder, preservative, stabilizer, flavor, etc., in aunit dosage form as called for by accepted pharmaceutical practice. Theamount of active substance in these compositions or preparations is suchthat a suitable dosage in the range indicated is obtained.

Illustrative of the adjuvants which can be incorporated in tablets,capsules and the like are the following: a binder such as gumtragacanth, acacia, corn starch or gelatin; an excipient such asmicrocrystalline cellulose, a disintegrating agent such as corn starch,a pregelatinzed starch, alginic acid and the like; a lubricant such asmagnesium stearate; a sweetening agent such as sucrose, lactose orsaccharin; a flavoring agent such as peppermint, oil of wintergreen orcherry. When the dosage unitform is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier such as fattyoil. Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets maybe coated with shellac, sugar or both. A syrup or elixir may contain theactive compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice by dissolving or suspending theactive substance in a vehicle such as water for injection, a naturallyoccuring vegetable oil like sesame oil, coconut oil, peanut oil,cottonseed oil, etc., or a synthetic fatty vehicle like ethyl oleate orthe like. Buffers, preservatives, antioxidants and the like can beincorporated as required.

RECEPTOR BINDING ASSAY USING RABBIT AORTAE MEMBRANE PREPARATION

Three frozen rabbit aortae (obtained from Pel-Freeze Biologicals) weresuspended in 5 mM Tris-0.25M Sucrose, pH 7.4 buffer (50 ml) homogenized,and then centrifuged. The mixture was filtered through a cheesecloth andthe supernatant was centrifuged for 30 minutes at 20,000 rpm at 4° C.The pellet thus obtained was resuspended in 30 ml of 50 mM Tris-5 mMMgCl₂ buffer containing 0.2% Bovine Serum Albumin and 0.2 mg/mlBacitration and the suspension was used for 100 assay tubes. Samplestested for screening were done in duplicates. To the membranepreparation (0.25 ml) there was added ¹²⁵ I-Sar¹ Ile⁸ -angiotensin II[obtained from New England Nuclear] (10 ul; 20,000 cpm) with or withoutthe test sample and the mixture was incubated at 37° C. for 90 minutes.The mixture was then diluted with ice-cold 50 mM Tris-0.9% NaCl, pH 7.4(4 ml) and filtered through a glass fiber filter (GF/B Whatman 2.4"diameter). The filter was soaked in scintillation cocktail (10 ml) andcounted for radioactivity using Packard 2660 Tricarb liquidscintillation counter. The inhibitory concentration (IC₅₀) of potentialAII antagonist which gives 50% displacement of the total specificallybound ¹²⁵ I-Sar¹ Ile⁸ -angiotension II was presented as a measure of theefficacy of such compounds as AII antagonists.

Using the methodology described above, the compound of this inventionwas evaluated and was found to exhibit an activity of at least IC₅₀ <50μm thereby demonstrating and confirming the utility of the compound ofthis invention as an effective AII antagonist.

Preparation of Substrate Compound (II):5,7-Dimethyl-2-ethyl-3-(2'-(tetrazol-5-yl)biphen-4-yl)methyl-3H-imidazol]4,5-b[pyridineI. Preparation ofN-Triphenylmethyl-5-(4'-bromomethyl-biphen-2-yl)tetrazole Step 1:2-cyano-4'-methylbiphenyl

To a solution of p-bromotoluene (30 g) in dry ether (150 ml) at -78° C.,a solution of t-BuLi in pentane (1.7 M) (210 ml) was added slowly over aperiod of 1 hour and 30 minutes, using a dropping funnel. The bath wasthen removed and the mixture was stirred at room temperature for anadditional 2 hours. The content of the flask was then added slowly(using a cannula) at room temperature to a premixed solution of ZnCl₂ inether (1 M) (180 mL) and dry THF (360 mL). The mixture was stirred for 2hours at that temperature and the slurry was added (using a cannula) toa solution of 2-bromobenzonitrile (21.3 g) and NiCl₂ (Ph3P)₂ (2.1 g) indry THF (300 ml). The mixture, after stirring at room temperatureovernight (18 hours), was poured slowly with stirring into ice cold 0.5N HCl (1500 ml). The organic layer was separated, and the aqueous phasewas extracted with ether (3×300 ml). The combined organic layer waswashed with water, brine and then dried over MgSO₄. Removal of thesolvent gave the crude product as a semisolid mass (34 g). The materialwas purified on a silica gel flash column using ethylacetate/hexane(1:12) to give the desired nitrile as a low melting solid (28 g, 88%).NMR (CDCl₃) δ2.42 (s, 3H), 7.2-7.8 (m, 8H); FAB-MS: m/e 194 (M+H).

Step 2: Trimethylstannyl azide

To a concentrated solution of NaN₃ (40 g) in water (100 ml), a solutionof trimethylthin chloride (20 g) in dioxane (10 ml) was added in threeportions under vigorous stirring. An instantaneous precipitate formationwas observed. The mixture, after stirring overnight at room temperature,was filtered. The residue was washed with water, and dried under suctionand then in vacuo over P₂ O₅. Yield 18.7 g (81%), mp 132°-136° C.

Step 3: N-triphenylmethyl-5-(4'-bromomethyl-biphen-2-yl)tetrazole

The titled compound was prepared starting from 2-cyano-4'-methylbiphenyl(Step 1) as described in European patent Application EP 0,291,969.

II. Preparation of 5,7-Dimethyl-2-ethylimidazo[4,5-b]pyridine Step 1:2-Amino-4,6-dimethyl-3-nitropyridine

2-Amino-4,6-dimethylpyridine (10.0 g, 81.8 mmol) was added portion-wiseto 65 mL of H₂ SO₄ (conc. d=1.84) which was stirred (mechanical) at 0°C. After complete addition, the mixture was warmed to room temperatureuntil the mixture become homogeneous. The solution was then cooled to-10° C. and a pre-cooled (0° C.) mixture of concentrated HNO₃ (11.5 mL,d=1.40) and H₂ SO₄ (8.2 mL, d=1.84) was added at such a rate as not toraise the internal reaction temperature above -9° C. Ten minutes afterthe addition was complete this cooled (-10° C.) mixture was poured onto400 g of crushed ice. The resulting slurry was neutralized by theaddition of conc NH₄ OH (to pH 5.5) while cooling (ice bath). The solidwas isolated by filtration, and dried at room temperature to give 13.3 gof 2-nitramino-4,6-dimethylpyridine as a white solid.

To 75 mL of stirred conc H₂ SO₄ cooled to -5° C. (ice-salt bath) wasadded 4,6-dimethyl-2-nitraminopyridine (13.2 g, 79 mmol) portion-wise atsuch a rate as to maintain the internal temperature below -3° C. Themixture was warmed to 0° C. until homogeneous (30 minutes) at which timetlc (SiO₂, 1:1 EtOAc/hexanes on a NH₄ OH neutralized aliquot) indicatedthat the rearrangement was complete. The mixture was poured onto 400 gof crushed ice and the pH was adjusted to 5.5 by the addition ofconcentrated NH₄ OH. The resulting yellow slurry was cooled to 0° C.,filtered, washed with cold water (50 mL), and dried at room temperatureto give 10.32 g of a mixture of the title compound and the 5-nitroisomer in a 55:45 ratio (determined by ¹ H NMR). This mixture was useddirectly in Step 2.

Step 2: 5,7-Dimethyl-2-ethylimidazo[ 4,5-b]pyridine

To a mixture of 8.44 g of a 55:45 mixture of2-Amino-3-nitro-4,6-dimethylpyridine and2-Amino-4,6-dimethyl-5-nitropyridine in MeOH (1.2 L) was added 10% Pd/C(2.4 g). The reaction vessel was evacuated then purged with H₂ at 1 atm.and stirred vigorously for 18 hours. Filtration (celite), andconcentration gave 6.65 g of a mixture of2,3-diamino-4,6-dimethylpyridine and 2,5-diamino-4,6-dimethylpyridine asa dark solid. To 5.40 g (39.4 mmol) of this mixture was added propionicacid (8.80 mL, 118 mmol) followed by polyphosphoric acid (100 mL). Thisstirred mixture was heated to 90° C. for 3 hours then to 100° C. for 1hour. After the reaction was complete, the warm mixture was poured onto300 g of ice and the mixture was made basic with NH₄ OH. The mixture wasextracted (4×50 mL CH₂ Cl₂), dried (K₂ CO₃) and concentrated to give amixture of the title compound and4,6-dimethyl-2,5-bis(propionamido)pyridine. Purification (SiO₂, 5% MeOH/EtOAc) gave 1.66 g of the tilte compound as the slower elutingcomponent. ¹ H NMR (CD₃ OD, 300MHz) δ6.95 (s, 1H), 2.92 (q, 2H, J=7.8Hz), 2.54 (apparent s, 6H), 1.40 (t, 3H, J=7.8 Hz)

III.5,7-Dimethyl-2-ethyl-3-(2'-(tetrazol-5-yl)biphen-4-yl)methyl-3H-imidazo[4,5-b]-pyridinePart A

To a stirred suspension of NaH (17.2 mmol of an 80% dispersion) in drydimethylformamide (30 mL) at rt was added5.7-dimethyl-2-ethylimidazo[4,5-b]pyridine (1.51 g, 8.62 mmol) in oneportion. After 20 minutes, the mixture was cooled to 0° C. andN-triphenylmethyl-5-(4'-bromomethylbiphenyl-2-yl)-tetrazole (5.29 g,9.48 mmol) was added in one portion. The resulting mixture was warmed tort and stirred for 15 hours. The excess NaH was quenched with water andthe bulk of the DMF was removed in vacuo at 40°-50° C. The residue wasextracted with EtOAc from brine, dried (K₂ CO₃), and concentrated.Purification by flash chromatography (SiO₂, solvent gradient: 80%EtOAc/hexanes, 100% EtOAc) gave 4.25 g of5,7-dimethyl-2-ethyl-3-(2'-(N-triphenylmethyltetrazol-5-yl)biphen-4-yl)methyl-3H-imidazo[4,5-b]pyridine:¹ H NMR (300 MHz, CDCl₃) δ7.86 (dd, 1H, J=7, 2 Hz), 750-7.41 (m, 2H),7.36-721 (m, 10 H), 7.05 (d, 2 H, J=4.5 Hz), 6.95-6.89 (m, 7 H), 6.86(d, 2 H, J=4.5 Hz), 5.35 (s, 2H), 2.67 (q, 2 H, J=7.5 Hz), 2.65 (s, 3H), 2.58 (s, 3 H), 1.25 (t, 3 H, J=7.5 Hz).

Part B

To a stirred solution of the tritylprotected tetrazole (4.13 g, 6.33mmol) in CH₂ Cl₂ (40 mL) at room temperature was added 85% formic acid(60 mL). After 45 minutes, the mixture was concentrated and the residuewas purified by chromatography (SiO₂, 85:13.5:1.5 CHCl₃ -MeOH-NR₄ OH)followed by crystallization from 30 mL of MeOH to give 2.18 g (84%)solid: mp 156°-158° C. ¹ H NMR (300 MHz, CD₃ OH) δ7.68-7.61 (m, 2 H),7.57-7.50 (m, 2 H), 7.07 (apparent singlet, 4 H), 7.04 (s, 1H), 5.55 (s,2 H), 2.85 (Q, 2 H, J=7.5 Hz), 2.61 (s, 3 H), 2.58 (s, 3 H), 1.25 (t, 3H, J=7.5 Hz).

Anal. Calcd for C₂₄ H₂₃ N₇.0.25 H₂ O: C, 69.63; H, 5.72; N, 23,68.Found: C, 69.91; H, 5.73; N, 23.60.

The following examples are given for the purpose of illustrating thepresent invention and should not be construed as being limitations onthe scope or spirit of the instant invention.

EXAMPLE 1 Microorganism and Culture Conditions

A frozen vial (2.0 ml) of culture MA 6751 (ATCC No. 55043) was used toinoculate a 250 ml baffled shake flask containing 50 ml of an autoclaved(sterilized) seed medium consisting of (in units of grams/liter) dextrin10.0, dextrose 1.0, beef extract 3.0, ardamine PH (Yeast Products, Inc.)5.0, N-Z Amine type E 5.0, MgSO₄.7H₂ O 0.05, K₂ HPO₄ 0.3, and CaCO₃ 0.5.The pH of the seed medium was adjusted to 7.1 before autoclaving. Theseed was incubated in the seed medium at 27° C. for 24 hours on a rotaryshaker operating at 220 rpm. A 2.5 ml aliquot of the resulting seedmedium was used to inoculate each of two 250 ml non-baffled shakeflasks, each flask containing 50 ml of the following previouslyautoclaved (sterilized) transformation medium B.* Substrate compound(II) was added to each flask as an aqueous solution with pH of 8 toachieve a final concentration of 0.1 mg/ml. The shake flask contentswere subsequently incubated for 24 hours at 27° C. on a rotary shakeroperating at 220 rpm. The resultant broths were combined for isolationand purification.

Isolation and Purification Procedure for the Broth

The whole broth (105 ml) of transformation media B was acidified to pH2.0 and then extracted three times with methylene chloride (3×100 ml).Methylene chloride extracts were combined, dried over sodium sulfate,and concentrated under vacuum to an oily residue. The residue wasdissolved in methanol and subjected to high performance liquidchromatography (HPLC) purification.

HPLC was carried out on Whatman Partisil 10 ODS-3, 9.4 mm×25 cm columnat room temperature and monitored at 255 nm and 275 nm. The column wasdeveloped at 3 ml/min with linear gradient from 0.1% aqueous TFA-CH₃ CN,85:15 to 0.1% aqueous TFA-CH₃ CN, 15:85 in 60 minutes. The compound wascollected during repeated injections of the above described extract. Thecompound was visible at 255 nm and 275 nm. The fractions at retentiontime 24.5 minutes were pooled and evaporated to remove solvents to yield4.0 mg. of compound (I).

Characterization

Compound (I) of this invention was characterized via NMR spectrometryyielding the proton NMR spectrum of FIG. 1, which also contains theassigned molecular structure.

What is claimed:
 1. A compound of structural formula (I): ##STR7##
 2. Anantihypertensive pharmaceutical composition comprising a non-toxictherapeutically effective amount of the compound of claim 1 and apharmaceutically acceptable carrier.
 3. A method of treatinghypertension of congestive heart failure comprising the administrationof a non-toxic therapeutically effective amount of the compound of claim1 to a subject in need of such treatment.